Flow cytometry is a well-known fluorescent analysis technique, which can, for example, be used to detect and separate blood cells in a suspension according to their phenotypic properties. This can be achieved by staining the cells in the sample to be analysed for example with a monoclonal antibody specific to a phenotypic marker, which itself is coupled to a fluorescent dye. Different phenotypic cell surface markers can therefore be distinguished when dyes with different fluorescent wavelengths, e.g. green and red, are coupled to a number of different antibodies, each specific to a different marker.
To perform this analysis technique a Fluorescence Activated Cell Sorter, generally called FACS, is known, which is a complex device comprising optical and fluid-handling subsystems, which are usually assembled from discrete components. An example of such a FACS is disclosed in Roitt, Brostoff, Male, Immunology 5th edition, Mosby Publishers (1998), p385, and is shown in FIG. 1. In FIG. 1 a fluorescent-stained sample cell suspension 1 is introduced into a vibrating flow cell 2.
A cell flow 4 passing out of the flow cell 2 is encased in a sheath of buffer fluid 3, which has been separately introduced into the vibrating flow cell 2. The flow cell uses the principle of laminar flow and hydrodynamic focussing. A laminar flow of liquid, i.e. a flow that is non-turbulent, passing through a cylindrical tube will be subject to a viscous drag at the wall of the tube that leads to a higher flow velocity near the tube centre. The resulting velocity profile is that of a parabola. The so called Bernoulli effect associates such a differential velocity profile with a pressure gradient that points radially inwards from the tube wall, i.e. the pressure in the liquid flow decreases from the wall to the centre of the tube. This pressure gradient will move any particle in suspension in the flow into the centre of the flow where it will remain. To prevent blocking, a suspension of particles can be introduced into the tube through a wide bore which is surrounded concentrically by a larger bore containing sheath fluid. By constricting this coaxial flow whilst maintaining laminar flow a focused stream of particles can be obtained. If a suspension of discrete particles is introduced into the tube in this manner the particles will flow through the centre of the tube in sequence, aligned behind one another. The sheath fluid commonly used in flow cytometry is phosphate buffered saline solution or a similar electrolyte solution which can be charged electrically.
The flow 4 passing out of the flow cell 2 is illuminated by a laser 5 within an interrogation region. The emitted laser beam is scattered at the fluorescent-stained sample cells in the suspension in a characteristic manner according to the specific optical properties of the particles or analytes in the suspension and the fluorescent dyes used, to detect specific markers.
Scattered and fluorescent light passing from the cell flow and emerging from the interrogation region is collected by a light directing subsystem with a plurality of beam splitters 6, collimating and focussing lenses (not shown) and is directed to a detection subsystem with several detectors 7, 8, 9, 10. Detector 7 measures forward scatter of incident light from the laser, which allows for an estimation of the size of the cells in the flow 4 passing the laser 5. Similarly the granularity of cells can be detected, by collecting light scattered at a 90 degree angle with detector 8 after the light has been redirected by one of the beam splitters 6. Finally, detectors 9 and 10 detect, for example, red and green fluorescence, emitted by green and red fluorescent dyes, respectively, to identify surface markers present on the cells. This is achieved by collecting fluorescent light emitted in the same 90 degree path leading away from the flow 4 and by passing the light through a second beam splitter in the plurality of beam splitters 6 to illuminate the detectors 9 and 10. The detectors 9 and 10 are therefore designed to only detect light emitted in the green and red wavelength bands of interest, respectively.
Where possible, fluorescent dyes will be used that have a common excitation wavelength maximum that can be excited by a single light source and different emission wavelength maxima, such as in the green or red spectrum. Most commercial flow cytometry systems in use today will detect between 2 and 6 fluorescent wavelengths. The detectors themselves are in most cases photomultiplier tubes and the wavelength discrimination is normally achieved by inserting bandpass filters in the light path before the entry facet of the receiving tube. Because of the size of the individual components in the detection subsystem it is challenging to manage the spatial arrangement of all optical components when a system with a relatively large number of detectors is required. In such a situation, the lengths of different light paths from the flow cell to the respective detectors may vary substantially between different detectors. These constraints tend to limit the number of fluorescent wavelengths that are typically used in commercial applications today.
For the event that cell sorting may be required, the end of the flow cell 2 has a vibrating nozzle, which causes the flow 4 emanating from the flow cell into air to break up into droplets that usually contain no more than a single cell. These droplets, falling from the nozzle perpendicular towards the ground due to their initial velocity and gravity, enter into an analyte sorting subsystem, where they are subsequently passed through a charging collar 11, which applies a substantially uniform electrical charge to each droplet. When sorting is required, the data received by the detectors 7, 8, 9, 10 can be processed to steer electrostatic deflection plates 12 under computer control, which allows re-directing different cell populations in the flow 4 at different angles into different ones of a plurality of output sample tubes 13, according to the fluorescent and other optical properties detected.
In an alternative embodiment mechanical sorting is known wherein either the final receptacle collecting the flow emanating from the flow cell or its inlet is switched electromechanically in order to receive the relevant fraction of cells.
As mentioned before, typical commercial FACS devices in use today can distinguish between 2 and 6 fluorescent colours. At the same time they are bulky and expensive limiting their broad use in research and clinical practice. They require precision bulk optical and discrete mechanical components. At the same time, however, the requirement of researchers to distinguish a higher number of variables in analyte samples is increasing.
Cell sorters that detect a larger number of colours have been designed, but are not used in wide practice. This is mainly due to the fact that extending the existing bulk optical and fluidics technologies to higher numbers of colours that can be discriminated is very expensive and cumbersome.
There are also known first attempts of miniaturising FACS-devices. For example a conventional flow chamber of a FACS is replaced by microfluidic devices manufactured by micromachining technology like soft lithography. In a paper “A micorfabricated fluorescence-activated Cell Sorter” of Anne Y. Fu et al., Nature Biotech., Vol17, p. 1109 (November 1999) there is described a FACS for sorting various biological analytes. A cell sorting device is produced as a silicone chip with channels for a sample liquid. The chip is mounted on an inverted microscope, and fluorescence is excited near junctions of the channels with a focused laser beam generated by a bulk laser, which is directed onto the chip perpendicularly to the plane of the chip. The fluorescent emission is collected by the microscope and measured with a photomultiplier tube, which collects light emitted perpendicularly to the plane of the chip. The laser beam is focused perpendicular to the chip and a plurality of beam splitters, mirrors, etc. is necessary to guide the light.
U.S. Pat. No. 5,726,751 discloses a flow cytometer made of two components: a flow cytometer optical head and a disposable flow module. The flow module utilises a flow channel micromachined in a silicon wafer. The optical head comprises a laser to provide an illuminating beam and photodetectors. The laser and the photodetectors are arranged out of plane of the wafer, dependent on the angle of the analysing light beam, so that the photodetectors may collect the analysing beam.